The present invention is directed to the production of lactic acid directly from a bioconversion reactor ("bioreactor" or "fermentor") broth made in a process for the fermentation of saccharified grain mash or molasses by a homofermentative strain of microorganisms which convert fermentable free sugars in the broth to lactic acid. The term "broth" refers to a submerged culture fermentation broth in a fermentor, which broth contains the mash of ground grain which is saccharified before it is fermented to yield lactic acid as the predominant acid, and by-products. The term "fermentable free sugars" refers to pentoses and hexoses, but mainly hexoses, present in jet-cooked and saccharified grain mash. By "lactic acid" we refer to all forms of lactic acid molecular weight ("mol wt" for brevity) 90.08, namely `levorotatory` L(+), `dextrorotatory` D(-), or racemic L(+)D(-) lactic acid, and reference to lactic acid herein refers to the foregoing individually and severally, unless stated otherwise.
The problem to be solved is: how to produce a high yield of lactic acid from the fermentor broth of a commodity grain until essentially all nutrient is used up, and to make the separation of essentially pure lactic acid economically feasible without precipitation of a salt of lactic acid (e.g. calcium lactate). The term "essentially pure" refers to recovered lactic acid having less than 5% by wt of low mol wt contaminants produced during a fermentation even if "corn thin stillage" ("thin stillage" for brevity) or "steep water" is used as a nutrient, the contamination being due to low mol wt compounds such as byproduct acetic acid and glycerol which are rejected together in reverse osmosis ("RO") modules. If neither thin stillage nor steep water is used, lactic acid produced may be 95% pure or higher.
The process of this invention is especially beneficial if it is operated in conjunction-with a fermentation plant producing ethanol from corn or sweet sorghum. In such a plant, thin stillage and steep water are waste streams, which are found to provide an exceptionally nutritious feed supplement for Lactobacilli which ingest the fermentable sugars present in corn mash, molasses, tapioca, potatoes and the like. The live microorganisms convert the sugars to lactic acid.
Thin stillage and steep water are produced in the dry milling and wet milling processes, respectively, for the production of ethanol from saccharified corn or sweet sorghum. To our knowledge, no prior art fermentation of grain mash, specifically corn or sweet sorghum mash, to produce lactic acid, has used either thin stillage or steep water as a nutrient. Of course, if either is unavailable there would be no over-riding reason for exploring their value as nutrients in this process. We use a portion of either by-product, typically in a minor proportion (less than 50%) preferably from 5% to 35% by weight (wet basis) of the fermentor's contents, in fermentations in which there is no objection to the small amount of glycerol introduced with the thin stillage. All percentages by weight (wt) given herein are on a wet basis unless specifically stated otherwise.
This process uses conventional unit operations which have been found, in combination, to be surprisingly result-effective. The process may be operated either in a "batch mode" with a batch fermentor; or, in a "continuous mode" with a cell-recycle fermentor. The "continuous mode" which is preferred for the operation of a plant, is a combination of a "continuous batch mode" with a "continuous feed and bleed".
In the batch mode, there is no feed flow into the fermentor containing fermenting broth and saccharified mash, and the entire contents of the fermentor are allowed to ferment until the fermentation is substantially self-terminated. The batch process is favored for ease of operation and freedom from possible contamination of the broth because a fresh charge of mash is freshly inoculated for each fermentation. Preferably, plural batch fermentors are operated in tandem to avoid the lag time due to waiting repeatedly for a fermentation to be self-terminated in a single fermentor.
The continuous process is favored for economics since, except for the initial fermentation, there is no lag time for production of the lactic acid, and labor and other overhead costs are lower. In the "continuous batch mode" also known as the "modified batch mode" only the `retentate`, also termed `concentrate`, is returned to the fermentor while permeate containing the lactic acid is withdrawn, and fresh feed is continuously supplied to the fermentor, to maintain a constant level in the fermentor. During continuous operation, returning retentate to the fermentor gradually increases the concentration of rejected undissolved solids in it, and a portion of the contents of the fermentor are purged from time to time. Because the pH is controlled within a narrow range in this novel process, returning only the retentate results in the operation of a cell-recycle fermentor. The concentration of cells in the fermentor is increased, but that of lactic acid is not permitted to exceed 3% by wt (without pH control), above which cells known today have difficulty staying alive, and do not grow noticeably. Though the principle of using a cell-recycle fermentor to increase cell concentration, that is CFU/ml (colony-forming units/ml), is known, one could not predict the effects of using the principle under the conditions of this process. Because of the effects of doing so, described in detail hereafter, the production and recovery of lactic acid is unexpectedly highly efficient.
Thin stillage typically contains a substantial concentration of water-insoluble ("suspended") solids, as well as "dissolved" solids, only the solids larger than about 15 .mu.m (micrometers or microns) in nominal diameter having been removed. If thin stillage is not used as a feed component to the fermentor, comparable nutrients are typically required to be added to maintain the same healthy state of cell growth. The disadvantage of using thin stillage is that the glycerol therein stays with the lactic and acetic acids, decreasing the purity of the lactic acid. Since thin stillage is discharged from a centrifuge which separates relatively large, water-insoluble solids in "whole still-age" leaving the thin stillage, some solids larger than 500 .mu.m are likely to remain with the thin stillage and, for reliability, it is desirable to prefilter broth withdrawn from the fermentor through a fine screen in the mesh size range from about 250 .mu.m-500 .mu.m, preferably 250 .mu.m mesh size, to remove the "heavy solids" from the mash before introducing the broth into an ultrafiltration ("UF") zone containing module(s) of ultraporous tubular membranes with pores no greater than 0.375 .mu.m.
If it is desired to separate the glycerol from the lactic acid in the product stream obtained as the retentate from a RO (reverse osmosis) zone, the glycerol is separated by known separation means, for example, ion exchange resins, as is the acetic acid, if it is to be separated. Where glycerol is not detrimental, it is left in the lactic acid, irrespective of whether the acetic acid is separated.
In the parent application, lactic acid was recovered as a by-product of a fermentation process in which corn was used to produce ethanol. Evolving economics, now skewed by a heightened awareness of the benefits of using a renewable resource combined with a topical demand for lactic acid, led to exploring the fermentation of corn and sweet sorghum, specifically, to yield lactic acid as the main acid product.
Lactic acid is not nearly as large a commodity bio-chemical as it would be, if only it could be produced more economically than it is today. It is now used in specialty chemicals, as a food acidulant and flavoring, and in pharmaceutical products, but several times as much could be used in textiles, and for other industrial applications such as for biodegradable synthetic resinous materials if lactic acid was readily available in volume. To date, the yearly increase in production of lactic acid has been due to its use in food and pharmaceutical products but it is expected that 0.5 billion Kg of lactic acid will now be used.
Lactic acid is now manufactured by one of several synthetic and fermentative methods, for the reason that sources of free sugars, such as molasses, potatoes or starch, for mono- and disaccharides, are not only less costly to purchase (alas, not "free") but also are renewable. Further the processing costs for fermentation processes are lower than those for synthetic processes, particularly those based on petroleum-based raw materials. Free sugars include monosaccharides such as glucose, fructose, galactose, and disaccharides include sucrose and lactose.
Certain homofermentative strains producing essentially only lactic acid are preferred, such as Lactobacillus delbrueckii, L. casei, L. xylosus, L. plantarum, L. acidophilus, and L. bulgaricus. The first and second strains, namely L. delbrueckii and L. casei consume free 6-C sugars in corn mash and sweet sorghum mash such as glucose, sucrose, and fructose, but do not consume lactose. Other species consume lactose and galactose in addition to the other sugars and the choice of species, or a mixture of species, depends upon the particular raw material to be used. Whichever species, or mixture of species, chosen for use, the fermentation product will contain a substantial concentration of suspended solids, as well as "dissolved" solids.
In the past, recognizing the economic attraction of using a fermentation process for lactic acid, those skilled in the art have used various types of processes involving solvent extraction, adsorption and desorption, electrodialysis and combinations of the foregoing to arrive at an economically effective process. See Adsorptive Purification of Carboxylic Acids by Elizabeth E. Ernst and Donald W. McQuigg, Paper #5ae at the AIChE 1992 National Meeting, Separations Division preprints, First Topical Conference on Separation Technologies: New Developments and Opportunities, Nov. 2-6, 1992, Miami Beach, Fla. The relatively high price of lactic acid in the marketplace is evidence that producing lactic acid in volume, is difficult. It was therefore particularly noteworthy that what appeared to be the simplest and most straightforward separation process, namely multiple filtrations in series, was bypassed.
This reluctance to utilize multiple filtrations may have been inoculated by experiments reported in an article titled "Lactic Acid Production by Electrolysis Fermentation Using Immobilized Growing Cells" by Hongo, Nomura and Iwahara in Biotechnol. & Bioeng., 30, 788-793 (Oct 1987) in which they used a broth containing cells in membrane-equipped electrodialysis tanks and found that the membranes were fouled despite the cells being dead. Though it was known that cells could be removed in a "UF" zone, Datta in U.S. Pat. No. 4,885,247 deemed the process was impractical because removing cells by UF is expensive, particularly since the concentration of lactic acid in the fermented broth was generally no higher than 10% by weight on a wet basis (see e.g. U.S. Pat. No. 5,068,418 to S. Kulprathipanja, Table, col 3, line 60). Datta did not comment on the problem of maintaining live cells under relatively high fluid pressures and high shear energy to which the broth is subjected in a conventional pump which pumps the broth to be ultrafiltered.
Long before Datta's disclosure, it was known that acontinuous, high productivity, cell-recycle, activated sludge digester as disclosed in U.S. Pat. No. 3,472,765 to Budd et al, could be used to reduce the cost of bioconversion of sewage by increasing volumetric productivity based on continuously separating biomass from the contents of the digester and recycling the biomass to the digester, if the appropriate membranes could be provided to make the necessary separation. However, despite the fact that Pseudomonas cells predominantly present in activated sludge in the size range from 1 .mu.m to 2 .mu.m are easier to separate from the water than Lactobacilli which are smaller (about 1 .mu.m long, 0.5 .mu.m in diameter), Budd et al teach directly flowing the withdrawn activated sludge to a RO and UF membrane from which the retentate is recycled to the digester. The RO membrane would, of course, also provide the UF function, so that this UF function would be redundant.
There is no indication whether the membranes would be blinded or damaged under specified conditions, and, if not, the conditions under which they would not be. Nor did Budd et al consider that only an insignificantly small fraction, if any, of live cells leaving the digester might survive the pressure required to produce any permeate through any UF or RO membrane. Clearly increasing the concentration of dead cells in the digester at the expense of those left alive, is not calculated to improve the efficiency of the digestion process.
The '765 patent gives no consideration to criteria which would govern the acceptable performance of the UF or RO membranes, so that one can derive no suggestion that only tubular UF membranes are likely to give acceptable results, and such results would be derived only if the activated sludge leaving the reactor was to be pumped over the tubes at a velocity in excess of 3 m/sec, preferably from 5-10 m/sec, but not high enough to kill more than 25% of living cells in the stream being pumped, preferably no faster than 30 m/sec, at relatively low pressure.
Specifically, since the '765 patent expressly teaches that the contents of the activated sludge fermentor are pumped through a withdrawal conduit to a housing having a flat plate RO membrane "selected so that they perform a reverse-osmosis and ultrafiltration separation operation wherein the biological life is retained on the feed side of the membrane", (see col 3, lines 68-70) without any suggestion that a UF separation be carried out first, it is clear Budd et al did not recognize the criticality of dealing with the solids in the broth, first, with a tubular UF filter, then, filtering the UF permeate in a RO filter, irrespective of the RO filter's configuration. Nor did they recognize that a preferred combination of a tubular UF and a NF filter of any configuration, would reliably produce a permeate essentially free of suspended solids, making it practical to filter the permeate from the NF filter in a RO zone. Further, though Budd et al sought a permeate essentially free of suspended solids they chose a UF or RO membrane to provide it, and did not recognize that only the latter (RO) could. Moreover, their choice of a UF membrane negates the probability that they could have appreciated the criticality of the relationship of cell size to pore size, and suggests they neither knew nor experienced the severity of the problem which existed. Thus, they found no need for any intermediate separations. In contrast, since the thrust of this invention is to provide lactic acid in volume, efficiently, it not only requires that there be multiple separations, but also that the pore size of the first UF ultraporous zone be critically related, that is, "cell-matched" to the diameter of the cells, as defined herebelow.
Still further, Budd et al failed to recognize that the pitfalls of filtering the cell-containing sludge, could only be avoided by using a cross-flow tubular UF means having a pore size in the range from about one-fourth (0.25), preferably one-half (0.5), the average nominal diameter of a cell, up to no more than three-fourths (0.75) that diameter. It is only with this combination of "cell-matched" ultraporous means in cross-flow filtration that our process gives acceptable recovery of cells and permeate. Even if it had been appreciated that a stream essentially free of suspended solids had been sought for the RO zone, and Budd et al had used a membrane with very small pores, smaller than 0.1 .mu.m, the filtration would be too inefficient to be practical.
The term "cross-flow" entails the flow of three streams--feed, permeate and retentate. In contrast, a "dead end" or "depth" filter has only two streams--feed and filtrate (or permeate). In cross-flow, feed flows through membrane channels, either parallel (or tangential) to the membrane surface, and is separated into a retentate (and/or recycle) stream and a permeate stream. The recycle stream retains all the particles and large molecules rejected by the membrane. The feed/recycle stream mixture flows through filter channels and may be totally recycled to the membrane module in which the recycle is generated, or partially removed from the system as reject (retentate). The key to maintaining an efficient membrane is to flow broth, at a sufficiently high velocity, parallel to the membrane surface to create shear forces and/or turbulence to sweep away accumulating particles rejected by the membrane.
To date, a favored method for the separation of lactic acid is to precipitate the calcium salt. The resulting calcium lactate is filtered to remove heavy metals and some organic impurities. The regenerated lactic acid is separated from precipitated CaSO.sub.4 (say), e.g., by filtration, and the resulting crude lactic acid further purified by carbon treatment and sodium ferrocyanide to remove additional organic impurities and heavy metals, respectively. After filtration, the lactic acid is contacted with an ion exchange resin to remove trace ions. It is immediately evident that this separation and purification process is complex and lactic acid purity in excess of 99% is difficult to obtain.
As indicated at the outset, it was the goal to replace the prior art process with an effective membranous separation of lactic acid from a suitable broth, which instigated the search for a "membrane solution" to the problem of separating lactic acid-efficiently. Such a separation was conceptually incited by a membranous separation of lactic acid from a fermentation broth to produce ethanol, described in the parent case. Except the broth is now generated with predominant strains of L. casei and L. delbrueckii and the like, which are gradually mutated by being recycled, to tolerate lower and lower pH levels in the range from pH 4.0 to 5.5, without sacrificing the production of lactic acid.
Because lactic acid is present in prior art broths in relatively low concentration, less than 8% on a weight basis (weight lactic acid / weight of broth or "w/w") even with pH control, recovering the lactic acid by filtration alone, has been such a technically unpromising and predictably unrewarding task, that to date, there has been no attempt, in a commercial process, to explore a filtration-only separation process. There is no record in the prior art of a suggestion as to how or why a filtration-only membrane process might be imminently operable; nor is there any suggestion that such a process will have the ability, economically to recover lactic acid from a fermented broth of grain.
Besides lactic acid among the dissolved solids, are numerous by-products since, generally, only the solids larger than 1 .mu.m in nominal diameter having been removed by microfiltration ("MF"). Dissolved solids include cell-compatible buffers and growth-enhancing nutrients, carbohydrates, amino acids, proteins and naturally occurring salts, and products of the bioconversion of sugars, all together referred to as "by-products". By "cell-compatible" buffer is meant one that improves production of lactic acid relative to an unbuffered broth, without stunting the growth of the active bacteria. The concentrations of byproducts ranges from about 100 ppm to 2 percent by weight of fermented broth, depending upon where in the process the concentration is measured. These by-products must be separated from the broth, to yield essentially pure lactic acid. To do so is neither simple nor inexpensive.
The reason for the failure to provide an economical process appears to have been a widespread misappraisal and misjudgment of the ability of a membrane means to make a critically important separation in the UF filtration step; and, the failure to realize that the relatively low mol wt of lactic acid relative to other by-products, fortuitously allows its separation and recovery from an aqueous stream containing proteins and relatively high mol wt (C.sub.10 -C.sub.24) fatty acids, provided the "right" membranes are used in the "right" combination.
Further, since it could be calculated that under the most desirable conditions achieved in the prior art, the overall permeate recovery of a fermentation broth in a UF, NF or RO module appeared to be unattractive, there was no motivation to probe the advantages of a process in which the broth was ultrafiltered, then nanofiltered, then RO filtered, under particularly specified conditions which provide at least 50% recovery in each step. There was little reason to suspect that such a membrane process could be economical. There was less reason to suspect that a membrane process could avoid or overcome the fouling problems endemic to such processes, particularly since it is well known that such problems are the basis for disproportionately large operating and maintenance costs.
In the context of this process it is important to note that the term UF refers to a membrane having pores no larger than 0.375 .mu.m, this being 0.75 times the .apprxeq.0.5 .mu.m diameter of a generally rod-shaped Lactobacillus, or 0.75.times.0.5 .mu.m=0.375 .mu.m and if one was to use a microfiltration (MF) membrane having a microporous pore size of 0.4 .mu.m, rod-like cells partially inserted end-wise in the pores, would effectively blind the pores and the microfiltration is inefficient. When so inserted, the trapped cells, or portions thereof, defy being dislodged even with back-pulsing the membrane tubes.
The pores in a conventional MF membrane are generally accepted as being in the size range from about &gt;0.2 .mu.m, ranging up to 10 .mu.m. A MF module is typically used to remove finely divided suspended solids much larger than cells, along with those cells which may be closely associated with the removed solids. The high recovery rate in our process is based, in the main, upon the discovery that a pore size no larger than 0.375 .mu.m, preferably 0.2 .mu.m, produces a cell-free permeate, and a membrane with this pore size resists blinding, fouling and damage. The productivity of a fermentation process with UF membranes having a smaller pore size than 0.1 .mu.m is inefficient and uneconomical.
A NF membrane being semipermeable and non-porous, provides a subsequent intermediate separation based both on mol wt and ionic charge. Mol wt cut-offs for non-ionized molecules are typically in the range from 150-1000 Daltons, referred to as "lights". For ions of the same mol wt, membrane rejections will increase progressively for ionic charges of 0, 1, 2, 3 etc. for a particular membrane because of increasing charge density (see "Nanofiltration Extends the Range of Membrane Filtration" by Peter Eriksson, Environmental Progress, Vol 7, No 1, pp 58-59, February 1988). The effectiveness of the NF semipermeable membrane means in this process derives from the presence of a large portion of the lactic acid in the UF permeate being present as lactate anions and H.sup.+ cations in equilibrium with non-ionized lactic acid.
A RO membrane is also semipermeable and non-porous, but requires an aqueous feed to be pumped to it at a pressure above the osmotic pressure of the dissolved substances in the water. Because an RO membrane can effectively remove low mol wt molecules&lt;150 Daltons, and also ions from water, RO membranes are commonly used to demineralize water (e.g. for pretreating boiler feedwater, and recovering potable water from brackish water or sea water). An RO membrane is ideally suited for making the separation of low mol wt lactic acid HOOC--CH(CH.sub.3)--OH=90, from higher mol wt components in the NF permeate.